Uses for androst-5-ene-3β, 17β-diol

ABSTRACT

Androst-5-ene-3beta,17beta diol is used to treat or reduce the likelihood of acquiring osteoporosis or menopausal symptoms, or other diseases affected by estrogen receptor activity, and for conditions which respond well to DHEA treatment, but where a higher ratio of estrogenic to androgenic effects is desired. Combination therapies are included, as are kits and pharmaceutical compositions for providing the active ingredients of claimed methods and combinations.

This application is a continuation-in-part of Ser. No. 09/096,286 filedJun. 11, 1998, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical compositions, kits andmethods for preventing and treating reduced or imbalanced concentrationsof sex steroids and conditions which can respond favorably to increasedactivity of androgens and/or estrogens. The invention utilizesandrost-5-ene-3β,17β-diol (hereinafter 5-DIOL) or compounds converted invivo to 5-DIOL

2. Description of the Related Art

5-DIOL is a compound biosynthesized from DHEA through the action ofreductive 17β-hydroxysteroid dehydrogenase (17β-HSD) and is a weakestrogen. It has an 85-fold lower affinity than 17β-estradiol (E₂) forthe estrogen receptor in rat anterior pituitary gland cytosol (Simardand Labrie, J. Steroid Biochem., 26: 539-546, 1987), further confirmingthe data obtained on the same parameter in human myometrial and breastcancer tissue (Kreitmann and Bayard, J. Steroid Biochem., 11: 1589-1595,1979; Adams et al., Cancer Res., 41: 4720-4926, 1981; Poulin and Labrie,Cancer Res., 46: 4933-4937, 1986).

At concentrations well within the range of the plasma levels found inadult women, 5-DIOL enhances cell proliferation and progesteronereceptor levels in human mammary tumor ZR-75-1 cells which lack3β-hydroxysteroid dehydrogenase/D5-D4 isomerase activity (Poulin andLabrie, Cancer Res., 46: 4933-4937, 1986) and increases theestrogen-dependent synthesis of the 52 kDa glycoprotein in MCF-7 cell(Adams et al., Cancer Res., is 41: 4720-4926, 1981).

In general, it is known that the serum levels of DHEA and DHEA-Sdecrease with age and correspondingly, that there is a dramaticreduction in the formation of androgens and estrogens in peripheraltarget tissues and a marked decrease in the biochemical and cellularfunctions induced by sex steroids. As a result, DHEA and DHEA-S havebeen used in the treatment of a variety of conditions which areassociated with decrease and/or imbalances in the levels of sexsteroids. Recently, we have found that the serum levels of 5-dioldecrease markedly with age.

Osteoporosis, a condition which affects both men and women, isassociated with a decrease in androgens and estrogens. Estrogens havebeen shown to decrease the rate of bone degradation while androgens havebeen shown to build bone mass.

Menopausal symptoms have also been associated with a loss of estrogens,and low dose estrogen therapy is commonly used in perimenopausal andmenopausal women to relieve vasomotor symptoms, urogenital atrophy,irritability, sleep problems, loss of energy, osteoporosis, and othersymptoms associated with menopause.

In addition, breast cancer, cardiovascular disease, and insulinresistance have been associated with decreased serum levels of DHEA andDHEA-S and both DHEA and DHEA-S have been suggested to prevent or treatthese conditions. DHEA has also been suggested to have beneficialeffects in the treatment and/or prevention of obesity, diabetes,atherosclerosis, chemically-induced breast, skin, and colon cancer,autoimmune diseases, Alzheimer's disease, loss of memory, aging and tosupport energy, muscle mass, and longevity. Uses of DHEA as well as thebenefits of androgen and estrogen therapy are discussed in InternationalPatent Publication WO 94/16709.

DHEA and DHEA-S have been suggested to be better for the treatment ofthese conditions than standard estrogen and androgen therapy since theaction of DHEA and DHEA-S is targeted to the tissues which can convertDHEA and/or DHEA-S to specific sex steroids. However, high doses of DHEAare required to get the necessary estrogenic and androgenic effects.Most importantly, the androgenic effects of DHEA are predominant andtherefore, for conditions in which a higher proportion of estrogens isdesired or where androgenic side effects are a problem, especially inwomen, the present invention permits a better proportion of estrogenicversus androgenic effects.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide pharmaceuticalcompositions and kits which include 5-DIOL or prodrugs converted theretoin vivo. In some embodiments, the pharmaceutical compositions consistessential of 5-DIOL.

It is also an object of the present invention to provide methods oftreating and preventing imbalances or reductions in the levels of sexsteroid hormones (androgens and/or estrogens) raising 5-DIOL levels in apatient in need of such treatment or prevention.

It is a further object of this invention to provide methods of ortreating or reducing the risk of onset of conditions which respondfavorably to estrogenic activity, including vaginal atrophy,hypogonadism, diminished libido, skin atrophy, urinary incontinence,lipid, and lipoprotein imbalance, atherosclerosis, cardiovasculardisease ana symptoms of menopause (hot flushes, sleep disorders,Alzheimer's disease, Parkinson's disease, mental disorders, depression,loss of memory) by administering 5-DIOL. it is a further object of thisinvention to provide methods of preventing or treating conditions whichrespond favorably to androgenic activity, including breast cancer,ovarian cancer, endometrial cancer, diminished libido, skin atrophy,skin dryness, osteoporosis and symptoms of menopause by administering5-DIOL. A number of diseases that are affected by sex steroids (e.g.osteoporosis) respond favorably to both androgens and estrogens.

A patient in need of treatment or reducing the risk of onset of a givendisease is one who has either been diagnosed with such disease or onewho is susceptible to acquiring such disease.

Except where otherwise noted or where apparent from context, dosagesherein refer to weight of active compounds unaffected by pharmaceuticalexcipients, diluents, carries or other ingredients, although suchadditional ingredients are desirably included, as shown in the examplesherein. Any dosage form (capsule, tablet, injection or the like)commonly used in the pharmaceutical industry is appropriate for useherein, and the terms “excipient,” “diluent” or “carrier” include suchnon-active ingredients as are typically included, together with activeingredients in such dosage forms in the industry. For example, typicalcapsules, pills, enteric coatings, solid or liquid diluents orexcipients, flavorants, preservatives, or the like are included.

The invention also includes use of an active agent in the manufacture ofa medicament for treatment of a disease specified herein as susceptibleto that agent, or one component of a combination in the manufacture of amedicament for treatment of a disease, where the treatment furtherincludes another component of the claimed combination therapy.

It is an additional object of this invention to provide novelcontraceptive methods which include administering 5-DIOL.

5-DIOL is a metabolite of dehydroepiandrosterone (DHEA). It has now beendiscovered that 5-DIOL has an unexpected variation (relative to DHEA) inits androgenic and estrogenic effects. In particular, 5-DIOL isfive-fold more estrogenic than DHEA while its androgenic activity isonly one- to two-fold higher than that of DHEA, thus giving anestrogenic to androgenic ratio of approximately 3.0 for 5-DIOL comparedwith DHEA. On the other hand, at higher doses, 5-DIOL produces maximaleffects less androgenic than DHEA. Thus, the relative estrogenic versusandrogenic effects of administering 5-DIOL lie more toward estrogeniceffect than does a corresponding dosage of DHEA. Therefore, 5-DIOL isparticularly useful in treating and preventing conditions involving lowlevels of sex steroids where the estrogen level, in particular, hasfallen (i.e. more so than the androgen level). Indeed the invention isuseful wherever an estrogenic effect is desired to a greater extent thanis an androgenic effect. As explained below, 5-DIOL may be administeredalone or in combination with other therapeutic agents such asantiestrogens, androgens, progestins, estrogens, DHEA, DHEA-sulfate,LHRH agonists or antagonists, inhibitors of 17β-hydroxysteroiddehydrogenase, aromatase inhibitors, inhibitors of gonadal sex steroidsecretion, as part of a combination therapy.

In the context of the invention, any prodrugs of 5-DIOL may be used inplace of 5-DIOL, including 5-DIOL-fatty acids, as these will alsoincrease the serum levels of 5-DIOL.

Other features and advantages of the present invention will becomeapparent from the following description of the invention which refers tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the uterineweight in ovariectomized rats, an estrogen-sensitive parameter. Intactanimals are used as additional controls. The compounds were dissolved in50% ethanol—50% propylene glycol and were administered in 0.1 ml on thedorsal skin area (2 cm×2 cm).

FIG. 2 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serumluteinizing hormone (LH) concentration in ovariectomized rats, a measureof androgenic and/or estrogenic effect. Intact animals are used asadditional controls. The compounds were dissolved in 50% ethanol—50%propylene glycol and were administered in 0.1 ml on the dorsal skin area(2 cm×2 cm).

FIG. 3 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serum DHEAconcentration in ovariectomized rats. Intact animals are used asadditional controls. The compounds were dissolved in 50% ethanol—50%propylene glycol and were administered in 0.1 ml on the dorsal skin area(2 cm×2 cm).

FIG. 4 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serum 5-DIOLconcentration in ovariectomized rats. Intact animals are used asadditional controls. The compounds were dissolved in 50% ethanol—50%propylene glycol and were administered in 0.1 ml on the dorsal skin area(2 cm×2 cm).

FIG. 5 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serumandrostenedione (4-dione) concentration in ovariectomized rats, ameasure of androgenic and/or estrogenic effect. Intact animals are usedas additional controls. The compounds were dissolved in 50% ethanol—50%propylene glycol and were administered in 0.1 ml on the dorsal skin area(2 cm×2 cm).

FIG. 6 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serumtestosterone concentration in ovariectomized rats. Intact animals areused as additional controls. The compounds were dissolved in 50%ethanol—50% propylene glycol and were administered in 0.1 ml on thedorsal ski. area (2 cm×2 cm).

FIG. 7 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the serumdihydrotestosterone (DHT) concentration in ovariectomized rats. Intactanimals are used as additional controls. The compounds were dissolved in50% ethanol—50% propylene glycol and were administered in 0.1 ml on thedorsal skin area (2 cm×2 cm).

FIG. 8 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the ventralprostate weight in orchiectomized rats, a measure of androgenic effect.Intact animals are used as additional controls.

FIG. 9 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the seminalvesicle weight in orchiectomized rats, a measure of androgenic effect.Intact animals are used as additional controls.

FIG. 10 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the concentrationof the mRNA encoding the C1 component of prostatic binding protein(PBP-C1) in the ventral prostate of orchiectomized rats, a measure ofandrogenic effect. Intact animals are used as additional controls.

FIG. 11 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered percutaneously twice daily for 7 days, on the mRNA encodingthe C3 component of prostatic binding protein (PBP-C3) in the ventralprostate of orchiectomized rats, a measure of androgenic effect. Intactanimals are used as additional controls.

FIG. 12 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered subcutaneously twice daily for 7 days, on the uterineweight in ovariectomized rats, a measure of estrogenic effect. Intactanimals are used as additional controls.

FIG. 13 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered subcutaneously twice daily for 7 days, on the serum LHconcentration in ovariectomized rats, a measure of androgenic and/orestrogenic effect. Intact animals are used as additional controls.

FIG. 14 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered subcutaneously twice daily for 7 days, on prostate weightin orchiectomized rats, a measured of androgenic effect. Intact animalsare used as additional controls.

FIG. 15 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered subcutaneously twice daily for 7 days, on seminal vesicleweight in orchiectomized rats, a measure of androgenic effect. Intactanimals are used as additional controls.

FIG. 16 is a graph of the effect of increasing doses of DHEA or 5-DIOL,administered subcutaneously twice daily for 7 days, on the serum LHconcentration in orchiectomized rats, a measure of androgenic and/orestrogenic effect. Intact animals are used as additional controls.

FIG. 17 shows the plasma concentration of DHEA (ng/mL) (Y axis infunction of time (X-axis) after a single oral absorption of prodrugs ofandros-5-ene-3β,17β-diol (150 μmol/rat) in male rats. In the box, AUC 24h of DHEA induced by these compounds is reported.

EM-760 dehydroepiandrosterone EM-900 androst-5-ene-3β,17β-diol EM-1304androst-5-ene-3β,17β-diol 3- acetate EM-1305-CSandrost-5-ene-3β,17β-diol diacetate EM-1397 androst-5-ene-3β,17β-diolacetate 17 benzoate EM-1400 androst-5-ene-3β,17β-diol dibenzoate EM-1410androst-5-ene-3β,17β-diol dipropionate EM-1474-Dandrost-5-ene-3β,17β-diol dihemisuccinate

FIG. 18 shows the plasma concentration of androst-5-ene-3β,17β-diol(ng/mL) (Y axis) in function of time (X-axis) after a single oralabsorption of prodrugs of androst-5-ene-3β,17β-diol (150 μmol/rat) inmale rats. In the box, AUC 24 h of androst-5-ene-3β,17β-diol induced bythese compounds is reported.

EM-760 dehydroepiandrosterone EM-900 androst-5-ene-3β,17β-diol EM-1304androst-5-ene-3β,17β-diol 3- acetate EM-1305-CSandrost-5-ene-3β,17β-diol diacetate EM-1397 androst-5-ene-3β,17β-diolacetate 17 benzoate EM-1400 androst-5-ene-3β,17β-diol dibenzoate EM-1410androst-5-ene-3β,17β-diol dipropionate EM-1474-Dandrost-5-ene-3β,17β-diol dihemisuccinate

In Vivo Assays of Bioavailability of the Prodrugs ofandrost-5-ene-3β,17β-diol

1) Principle

The assays of the bioavailability of prodrugs ofandrost-5-ene-3β,17β-diol were performed in male Sprague Dawley rats bymeasuring the plasma concentrations of the compounds after single oraladministration of the compounds.

2) Animals and Treatment

Male Sprague-Dawley rats [Crl:CD(SB)Br] weighing 275-350 a were obtainedfrom Charles-River Canada Inc. and housed 2 per cage during theacclimation period and individually during the study period. The animalswere maintained under a regimen of 12 hours light; 12 hours dark (lightson at 08:00). Animals received certified Rodent feed (Lab Diet # 5002,pellets) and tap water ad libitum. Rats were fasted (access to wateronly) starting on the evening prior to dosing.

Each compound to be tested was administered to three animals as asuspension in 0.4% methylcellulose by oral gavage at a dose of 150μmol/rat. One blood sample of ˜0.7 ml was collected from the jugularvein of rats under Isoflurane-induced anesthesia at 1, 2, 3, 4 and 7hours post-gavage. Blood samples were immediately transferred into arefrigerated 0.75 ml Microtainer containing EDTA and kept in anice-water bath until centrifugation at 3000 rpm for 10 minutes. Plasmaseparation was performed rapidly (less than 50 minutes) after bloodcollection). One aliquot of 0.25 ml of plasma was then transferred intoa borosilicate tube (3×100) and was rapidly frozen on dry-ice. Plasmasamples were kept at ˜80° C. until measurement of plasma concentrationof the sex steroid or sex steroid precursors by GC-MS.

DETAILED DESCRIPTION OF THE INVENTION

It is believed that 5-DIOL may be used, in accordance with theinvention, for the treatment of any disease known to respond favorablyto treatment with DHEA with the added benefits related to its morefavorable ratio of estrogenic versus androgenic activities and its lowermaximal androgenic activity relative to DHEA. Administering 5-DIOLdirectly in accordance with the invention, has a number of advantagesover administering DHEA, as discussed herein.

Applicant has discovered that 5-DIOL produces significantly differentandrogenic and estrogenic effects than does DHEA. In particular, 5-DIOLis shown to produce less potential androgenic or masculinizing effectsfor a given production of estrogenic effects than does DHEA. Therefore,5-DIOL is particularly beneficial in treating conditions which requireestrogenic activity with minimal androgenic activity. In fact, aftermenopause, women have a deficit of both androgens and estrogens althoughthe ratio of estrogen/androgen is lower than before menopause. Womenthus require a more favorable estrogenic-androgenic ratio to compensatethe loss of estradiol secretion by the ovaries. DHEA cannot compensatefor this ovarian estrogenic deficit but will only replace the loweredsecretion of DHEA-S and DHEA by the adrenals.

In particular, the production of the active androgen DHT and itsprecursor 4-dione by administering DHEA ranged, for increasing dosages,from 30% to 125% greater than obtained with 5-DIOL administration. Onthe other hand, a 53% greater production of testosterone was obtainedwith DHEA than by administering 5-DIOL. 4-dione is a steroid which isitself a weak androgen but is particularly efficiently transformedintracellularly into the more potent androgens testosterone, and DHT.The data shows that the maximum level of serum DHT reached after 5-DIOLwas less than half of the maximum level reached with DHEA, and that at arelatively low dosage levels, the androgenic effect of 5-DIOL reaches aplateau of maximum activity much lower than that achieved by DHEA.

FIGS. 10 and 11 confirm the relatively higher level of androgenicactivity of DHEA as compared to the activity of 5-DIOL. The data shows,depending on dosage, that DHEA is two to five times more potent than5-DIOL in stimulating prostate binding protein-C1 (PBP-C1) and prostatebinding protein-C3 (PBP-C3) mRNA levels. The levels or PBP-C1 and PBP-C3mRNA are particularly sensitive parameters of androgenic action and theconcentration of the RNAs encoding these proteins is regulated byandrogens. Measurements of PBP-C1 and PBP-C3 mRNA levels are indicativeof the androgenic activity since androgens act at the transcriptionallevel to increase steady skate levels of the mRNAs encoding the subunitcomponents of PBP.

Because both DHEA and 5-DIOL administered percutaneously have almostidentical effects on estrogenic parameters at a given dosage, DHEAproduces two to three times more androgenic activity than 5-DIOL whereequivalent estrogenic effects are provided.

It has also been found that a manor difference between DHEA and 5-DIOLis that oxidative 17β-HSD(s) which transform(s) 5-DIOL into DHEA has anextremely low level of activity after systemic administration of 5-DIOL(see FIGS. 3 and 4). On the other hand, the similar levels of serum5-DIOL following administration of DHEA and 5-DIOL indicate that thereductive 17β-HSD has a relatively high level of activity. In addition,since no significant DHEA appears available to be transformed directlyinto 4-dione by 3β-HSD, following 5-DIOL administration, androstenedionemust derive rom the oxidation of testosterone. This is in agreement withthe fact that levels of serum 4-dione, a highly efficient precursor ofandrogens, are much lower after 5-DIOL treatment than after DHEAtreatment.

Without intending to be bound by theory, it is believed that one of thereasons DHEA produces a different androgenic response than does 5-DIOL(at equal dosage) is that DHEA and 5-DIOL are metabolized differently.5-DIOL's metabolism (more so than DHEA's metabolism) leads to lessformation and/or more inactivation of certain steroids (e.g.testosterone and DHT) before they can exert androgenic activity. Thus,5-DIOL leads to lower exposure to these potent androgens than does anequal dose of DHEA where the oxidized steroids predominate as areservoir for formation of the active androgens.

Further, as shown in FIGS. 14 and 15, where both compounds wereadministered by the subcutaneous route for an assessment or maximalbioavailability, the stimulation of androgen-sensitive parameters by5-DIOL reached a plateau at a lower value than achieved with DHEA. It isthus clear that the difference of maximal androgenic activity of DHEAand 5-DIOL observed after percutaneous administration of the twocompounds (FIGS. 8, 9, 10, and 11) is not due to different rates ofabsorption through the skin.

In general, the estrogenic activities of 5-DIOL under conditions ofmaximal bioavailability were shown to be greater than those of the samedoses of DHEA. In fact, after subcutaneous administration, 5-DIOL isabout times more potent than DHEA to stimulate uterine growth.Therefore, as discussed above, at a dosage where the estrogenic effectsare the same, the androgenic effects produced by 5-DIOL will besignificantly lower than the effects produced by DHEA. Moreover, theandrogenic effects of 5-DIOL reach a plateau of maximal effect which issignificantly lower than the maximal stimulation achieved with DHEA.

As will be discussed in detail below, 5-DIOL can be administered totreat or prevent conditions in patients who have insufficient levels orimbalanced concentrations of sex steroids, namely androgens and/orestrogens. In particular, 5-DIOL is believed useful in treating andreducing risk of acquiring conditions which respond favorably toestrogenic activity and in which lower androgenic activity than providedby DHEA is desired. Because 5-DIOL is metabolized qualitatively to thesame sex steroids as is DHEA, 5-DIOL may be used for any purpose thatresponds favorably to DHEA, with the benefit of different androgenicversus estrogenic effects than would result from using DHEA. Where thedesired estrogen/androgen ratio lies between what is achievable withDHEA versus 5-DIOL, he a mixture of DHEA and 5-DIOL may be used toprovide the desired ratio.

Individuals who will benefit from treatment with 5-DIOL include allthose suffering from conditions treatable with DHEA, including thosewith abnormally low levels of 5-DIOL, estrogen, or androgen. Reducingthe risk of acquiring such conditions is also possible and therecommended 5-DIOL dosage and target serum levels is the same as for thetherapeutic uses of 5-DIOL herein. Individuals who could benefit fromthe invention can be identified by measuring serum levels of 5-DIOL,DHEA, sex steroids and their metabolizes (especially androsteroneglucuronide and androstane-3a,17β-diol glucuronide for androgens andestrone-sulfate and estradiol-sulfate for estrogens) as described byBeélanger et al., in Steroid Formation, Degradation and Action inPeripheral, Normal and Neoplastic tissues (H. Bradlow, L. Castagnetta,S. d'Aquino, L. Gogliotti, eds) Ann. N.Y. Acad. Sci. 586: 93-100, 1990.Serum IGF-1 levels can be measured as described (Furlanetto et al., J.Clin. Invest. 60: 648, 1977).

In accordance with one aspect of the invention, once the deficiency orimbalance is determined, 5-DIOL is preferably administered at a dosagesufficient to raise and maintain serum 5-DIOL, concentrations up to 3times above the normal range of young adults. Serum Concentrations of5-DIOL between 4.0 nM and 10 nM for women and 10 to 20 nM for men arepreferred, e.g. 7.0 nM for women and 15 nM for men. Naturally, theattending clinician may raise or lower dosage based on patient responsewhich may vary significantly. Intermittent or continuous administrationof a progestin (e.g. medroxyprogesterone acetate, 5-10 mg/day orally)may alleviate possible unwanted side effects on the endometrium of the5-DIOL treatment in premenopausal women.

In some preferred embodiments, serum concentration is between 4.0 and7.0 or between 7.0 and 15 nM for women and men, respectively. However,for purposes of contraception or for prevention of ovarian or uterinecancer, concentrations up to 15 nM (e.g. between 10 and 13) may bepreferred for women. For contraception, an estrogen may be added (e.g.estradiol giving serum estradiol levels between 50 and 200 nanograms perliter), and an added progestin may be particularly appropriate.Preferred dosages discussed herein may be increased as appropriate toachieve desired serum concentrations, e.g. with variations forindividuals patient response as monitored by the attending clinician.

When 5-DIOL is administered by the percutaneous or transmucosaltechnique, the delivered dosage may be raised or lowered in known ways,i.e. by altering the location to which the lotion, ointment, cream, gelor patch is applied by altering the size of the surface area to which itis applied, by altering the concentration of the active ingredient, orby altering the vehicle or carrier. For example, increasing the surfacearea will normally increase the dosage of active ingredient delivered ifthe concentration of active ingredient remains constant. In the samemanner, dosage delivered increases with increased concentration ofactive ingredient in the delivery base, and decreases with decreasedconcentration. Dosage delivered into the bloodstream also varies in aknown manner with respect to the body region at which the transdermalpenetration system is applied to the skin. Changing the vehicle orcarrier can also alter the delivered dosage in known ways.

Preferably, serum 5-DIOL concentration is measured before treatmentbegins, and a dosage is selected to quickly raise serum 7-DIOLconcentration to the preferred target range between 4.0 and 10 in forwomen and 10 to 20 nM for men. Subsequently, the patient is monitoredboth symptomatologically and by circulating 5-DIOL or sex steroidmetabolite concentrations to verify that the desired serum concentrationand symptomatic relief have been obtained. 5-DIOL is then maintained ata constant concentration in the circulation. For a typicalpostmenopausal patient, for example, this dosage is the equivalent ofapplication of 400 mg of the 5-DIOL, as part of a 10 percent compositionin 50% ethanol—50% propylene glycol, to a 200 square centimeter area ofthe abdomen or thighs two times daily per 50 kg of body weight. If oraladministration is chosen, 500 mg should be administered twice daily per50 kg of body weight.

As used in the invention, 5-DIOL may be administered with or withoutadditional carrier or diluent by the oral route but requires anadditional carrier or diluent when administered by the percutaneous ortransmucosal route. In a pharmaceutical composition for oraladministration, 5-DIOL is preferably present in a concentration between5 and 99% by weight relative to total weight of the composition, morepreferably between 50 and 99 percent, especially between 80 and 99percent.

When prepared for percutaneous administration, 5-DIOL is preferablypresent in a concentration between 2 and 20% by weight relative to thetotal weight of the composition, more preferably between 5 and 15%,especially between 5 and 10%.

The 5-DIOL active ingredient may be obtained from Steraloids Inc. (P.O.Box 310, Wilton, N.H., 03086, USA). Preferred 5-diol prodrugs are thetwo compounds set forth below, commercially available from SteraloidsInc.

5-DIOL may be administered alone or may be administered in combinationwith other active compounds, such as antiestrogens, progestins,androgens, estrogens, DHEA or DHEA-S, inhibitors of 17β-Hydroxysteroiddehydrogenase, aromatase inhibitors, LHRH agonists or antagonists andother inhibitors of gonadal steroid secretion. Both DHEA and 5-DIOL aremetabolized to androgens and estrogens, but in different proportions. Inaddition, 5-DIOL possesses weak intrinsic estrogenic activity. That, inconjunction with the different action DHEA or 5-DIOL have on theandrogen or estrogen receptor, is believed to impart the differentratios of androgenic versus estrogenic activity provided by 5-DIOLversus DHEA. It is not necessary to utilize only DHEA'sandrogen/estrogen ratio or only 5-DIOL's androgen/estrogen ratio. DHEAand 5-DIOL may be used together to provide an optimally effectiveandrogenic/estrogenic response ratio that is between the ratio for DHEAand the ratio for 5-DIOL. The relative amounts of DHEA versus 5-DIOL maybe varied depending on whether the desired androgen/estrogen responseratio lies closest to that of DHEA or to that of 5-DIOL.

For the treatment of breast cancer, endometrial cancer, ovarian cancer,endometriosis or other estrogen-sensitive disease requiring blockade ofestrogen formation and/or action, at least one of the following, namelyan antiestrogen, an aromatase inhibitor, an androgenic compound, aprogestin, an LHRH agonist or antagonist or another inhibitor of gonadalsex steroid secretion, an inhibitor of 17β-hydroxysteroid dehydrogenaseactivity can be used in combination with 5-DIOL. 5-DIOL alone or withDHEA would provide the androgenic component required to stimulateandrogen-sensitive functions, particularly bone formation and inhibitionof androgen-sensitive cancer growth (e.g. breast and endometrialcancer). 5-DIOL could then be used alone or in combination with any ofthe compounds mentioned above useful in the combination. In some cases,DHEA in the absence of 5-DIOL could be used in combination with any ofthe compounds mentioned above.

In another embodiment, 5-DIOL is combined with an antiestrogen, which ispreferably EM-800 ((−)-7-pivaloyloxy-3-(4′-pivaloyloxyphenyl)-4-methyl-2(4′″-2(2-piperininoethoxy)phenyl)-2H-benzopyran), ICI 182,780(7a-[9-4(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-triene-3,17β-diol)or any other antiestrogen for the treatment of breast cancer endometrialcancer, ovarian cancer cardiovascular diseases, atherosclerosis, andother estrogen-sensitive diseases. Non-steroidal antiestrogens such asEM-800 tend to be selective estrogen receptor modulators which act asestrogen receptor antagonists in breast tissue, yet provideestrogen-like beneficial effects on cholesterol, lipids andatherosclerosis.

The dosage of 5-DIOL can vary. The blood level of 5-DIOL and of othersex steroids and their metabolites is indicative of the adequate dosagetaking into account individual variation in absorption, metabolism, andsensitivity of response. Preferably, the attending clinician will,especially at the beginning of treatment, monitor an individualpatient's overall response and serum levels of 5-DIOL (in comparison tothe preferred serum concentration discussed above), and monitor thepatient's overall response to treatment, adjusting dosages as necessarywhere a given patient's metabolism or reaction to treatment is atypical.For combination therapies, one approach would be to start treating with5-DIOL alone and to add the other compounds only if necessary. Fortreatment of breast cancer, endometrial cancer, ovarian cancer, andendometriosis, treatment with antiestrogen+5-DIOL orantiestrogen+5-DIOL+DHEA or antiestrogen+DHEA are startedsimultaneously. If DHEA androgen, progestin or estrogen is added,similar monitoring of overall serum levels, both of the activeingredients and androgenic or estrogenic metabolites is preferred duringearly stages of treatment and as judged useful by the physician at latertime intervals.

Treatment in accordance with the invention is suitable for indefinitecontinuation. It is expected that 5-DIOL treatment will usually simplymaintain this natural steroid within a range of 4 to 10 nM and 10 to 20nM serum concentration for women and men, respectively. Undesirable sideeffects rom sustained 5-DIOL treatment are expected to be either minimalor nonexistent. Avoiding unlikely side effects from sustained estrogenuse may be achieved in ways already known to the art, for example, byintermittent (or in some embodiments continuous) administration of aprogestin (e.g. medroxyprogesterone acetate) at a daily oral dose of 2to 10 mg. Any androgenic side effects should be minimal due to therelatively low androgenic effects of 5-DIOL and the already low levelsof DHEA in most patients undergoing the method of the invention (FIG.18).

In order to facilitate the combination therapy aspect of the invention,for any indication discussed herein, the invention contemplatespharmaceutical compositions which include both 5-DIOL and a second orsubsequent active compound(s) in a single composition for simultaneousadministration. The composition may be suitable for administration inany traditional manner including but not limited to oral administration,percutaneous administration or transmucosal administration. In otherembodiments, a kit is provided wherein the kin includes 5-DIOL and asecond compound in separate containers. Additional active compoundsdiscussed herein may also be included. In addition to other modes ofadministration, the second compound as well as 5-DIOL may also beadministered transdermally in accordance within the invention asdiscussed in more detail below. Thus, the kit may include appropriatematerials for transdermal administration, e.g., ointments, lotions,gels, creams, sustained release patches and the like. The same strategyapplies to the progestin, antiestrogen, androgen, estrogen, DHEA,DHEA-S, inhibitor or 17β-hydroxysteroid dehydrogenase, aromataseinhibitor or inhibitor of gonadal sex steroid secretion which can beadministered orally (or by infection for the LHRH agonist orantagonist).

Although, it is anticipated that in some circumstances 5-DIOL may beadministered by injection, this method is not favored. Since treatmentwith 5-DIOL will often be of prolonged and indefinite duration, repeateddelivery by injection is inconvenient.

It is believed that the preferred routes for therapeutic administrationof 5-DIOL are percutaneous, transmucosal or oral, since the discomfortand inconvenience of administering 5-DIOL by injection are avoided.

Any of a number of art-recognized transdermal penetration systems may beutilized for the delivery of 5-DIOL. For example, 5-DIOL may be preparedas part of an ointment, lotion, gel or cream for rubbing onto apatient's skin or mucosa. Ate active ingredient is preferably presentfrom approximately 5% to 20% by weight relative to the total weight ofthe pharmaceutical composition and more ore preferably is betweenapproximately 5 and 12% by weight. Alternatively, the active ingredientmay be placed into a transdermal patch having structures known in theart, for example, structures such as those set forth in E. P. Patent No.0279982.

When formulated as an ointment, lotion, gel, cream or the like, theactive compound is admixed with a suitable carrier which is compatiblewith human skin cream mucosa and which enhances transdermal ortransmucosal penetration of the compound Through the skin or mucosa.Suitable carriers are known in the art and include but are not limitedto Klucel HF and Glaxal base which is available from Glaxal CanadaLimited Company. Other suitable vehicles can be found in Koller andBurl, S.T.P. Pharma 3(2), 115-124, 1987. The carrier is preferably onein which the active ingredient(s) is(are) soluble at ambient temperatureat the concentration of active ingredient that is used. The carriershould have sufficient viscosity to maintain the precursor on alocalized area of skin or mucosa to which the composition has beenapplied, without running or evaporating for a time period sufficient topermit substantial penetration of the precursor through the localizedarea of skin or mucosa and into the bloodstream where it will causemeasurable and desired increase in serum 5-DIOL concentration. Thecarrier is typically a mixture of several components, e.g.pharmaceutically acceptable solvents and a thickening agent. A mixtureof organic and inorganic solvents can aid hydrophilic and lipophilicsolubility, e.g. water and an alcohol such as ethanol.

Desirably, the carrier is one which, formulated as 10% 5-DIOL and 90%carrier (by weight) and applied twice daily in an amount providing 100mg of 5-DIOL to the abdominal area, will elevate serum concentration of5-DIOL in a typical patient by at least 1.0 nM per 50 kg of body weightabove serum levels prior to treatment, and thereafter maintainrelatively constant serum levels of 5-DIOL.

The carrier may include various additives commonly used in ointments,lotions, gels, and creams and well known in the cosmetic and medicalarts. For example, fragrances, antioxidants, perfumes, gelling agents,thickening agents such as carboxymethylcellulose, surfactants,stabilizers, emollients, coloring agents and other similar agents may bepresent. When used to treat systemic diseases, the site of applicationon the skin is preferably changed periodically to avoid potential excesslocal concentration of steroids and possible overstimulation of the skinand sebaceous glands by androgenic metabolites of 5-DIOL.

5-DIOL or derivatives can also be administered, by the oral route, andmay be formulated with conventional pharmaceutical excipients, e.g.spray dried lactose and magnesium stearate into tablets or capsules ororal administration at concentrations providing easy dosage in a rangefrom 0.050 to 2.5 grams per day per 50 kg of body weight.

The active substance can be worked into tablets or dragee cores by beingmixed with solid, pulverulent carrier substances, such as sodiumcitrate, calcium carbonate or dicalcium phosphate, and binders such aspolyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly byadding also lubricants such as magnesium stearate, sodium laurylsulfate, “Carbowax” or polyethylene glycol. Of course, taste-improvingsubstances can be added in the case of oral administration forms. Theactive substance can be also administered in solid dispersion state inappropriate carriers. Such carriers may be chosen from the groupconsisting of polyethylene glycols of molecular weight varying from 1000to 20000 and polyvinylpyrrolidone (Povidone purchased from AmericanChemicals Ltd., Montréal, Canada).

As further forms, one can use plug capsules, e.g. of hard gelatin, aswell as closed soft-gelatin capsules comprising a softener orplasticized, e.g. glycerine. The plug capsules contain the activesubstance preferably in the form of granulate, e.g. in mixture withfilers, such as lactose, saccharose, mannitol, starches, such as potatostarch or amylopectin, cellulose derivatives or highly dispersed silicicacids. In solf-gelatin capsules, the active substance is preferablydissolved or suspended in suitable liquids, such as vegetable oils orliquid polyethylene glycols.

The concentration of active ingredients in the ointment, cream, gel orlotion is typically from about 2 to 20 percent preferably between 5 and15 percent and preferably between 5 and 10 percent (by weight relativeto the total weight of the lotion, cream, gel or ointment). Within thepreferred ranges, higher concentrations allow a suitable dosage to beachieved while applying the lotion, ointment, gel or cream to a lessersurface area of the skin than would be possible at lower concentrationsand allow more freedom in choosing the body parts to which the ointmentor lotion will be applied. For example, it is well known in the art thata compound which is capable of transdermal penetration normallypenetrates more efficiently at some points in the body than in others.For example, penetration is very efficient on the forearm considerablyless efficient on the palms.

The lotion, ointment, gel or cream should be thoroughly rubbed into theskin so that no excess is plainly visible, and the skin would not bewashed in that region until most of the transdermal penetration hasoccurred, preferably, at least 15 minutes and, more preferably, at least30 minutes after application.

A transdermal patch may be used to deliver 5-DIOL in accordance withknown techniques. It is typically applied for a much longer period, e.g.0.5 to 4 days, but typically contacts active ingredients to a smallersurface area, allowing a slow and constant delivery of activeingredient.

A number of transdermal drug delivery systems that have been developed,and are in use, are suitable for delivering the active ingredient of thepresent invention. The rate of release is typically controlled by amatrix diffusion, or by passage of the active ingredient through acontrolling membrane.

Mechanical aspects of transdermal devices are well known in the art, andare explained, for example, in U.S. Pat. Nos. 4,162,037, 5,154,922,5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343,4,064,654, 5,071,644, 5,071,657, the disclosures of which areincorporated herein by reference. Additional background is provided byEuropean Patent 0279982 and British Patent Application 2185187.

The device may be any of the general types known in the art includingadhesive matrix and reservoir-type transdermal delivery devices. Thedevice may include drug-containing matrixes incorporating fibers whichabsorb the active ingredient and/or carrier. In a reservoir-type device,the reservoir may be defined by a polymer membrane impermeable to thecarrier and to the active ingredient.

In a transdermal device, the device itself maintains active ingredientin contact with the desired localized skin surface. In such a device,the viscosity of the carrier or active ingredient is of less concernthan with a cream or gel. A solvent system for a transdermal device mayinclude, for example, oleic acid, linear alcohol lactate and dipropyleneglycol, or other solvent systems known in the art. The active ingredientmay be dissolved or suspended in the carrier.

For attachment to the skin, a transdermal patch may be mounted on asurgical adhesive tape having a hole punched in the middle. The adhesiveis preferably covered by a release liner to protect it prior to use.Typical material suitable for release includes polyethylene andpolyethylene-coated paper, and preferably silicone-coated for ease ofremoval. For applying the device, the release liner is simply peeledaway and the adhesive attached to the patient's skin. In U.S. Pat. No.4,135,480, the disclosure of which is incorporated by reference, Bannonet al. described an alternative device having a non-adhesive means forsecuring the device to the skin.

Except for the higher dosage indications noted above (e.g.contraception), the target serum concentration of 5-DIOL is comparableregardless of whether 5-DIOL is being used as part of a combinationtherapy for treatment of menopause or is being used (by itself or incombination with antiestrogens, androgens, progestins, estrogens andinhibitors of 17β-hydroxysteroid dehydrogenase, aromatase inhibitors,inhibitors of gonadal sex steroid formation, LHRH agonists orantagonists, DHEA and/or DHEA-S) for the treatment of cardiovasculardiseases, osteoporosis, skin deterioration, menopause, vaginal atrophy,urinary incontinence, uterine cancer, ovarian cancer, osteoporosis,endometriosis, hypogonadism or diminished libido in accordance with theinvention or for the treatment of any conditions related to decreases orimbalances in the levels of sex steroids, in particular 5-DIOL and itsmetabolites.

5-DIOL is particularly useful in treating conditions in which a minimalandrogenic effect is desired since the androgenic effects of 5-DIOL arelower than produced by DHEA for an equal estrogenic effect and themaximal androgenic effect achieved with 5-DIOL is lower than thatachieved with DHEA. 5-DIOL is especially preferred for the treatment ofconditions in women that respond to estrogen therapy (or therapy with anestrogen precursor, such as DHEA) since the androgenic action of 5-DIOLis lower than that of DHEA and therefore, the potential androgenic ormasculinizing effects are reduced, while the desired estrogenic activityis provided. Moreover, in postmenopausal women, in general, a greaterestrogenic/androgenic ratio than that provided by DHEA is required.

Since 5-DIOL is a natural source of estrogens and androgens and thesecretion of this compound markedly decreases during aging (FIG. 17),its replacement should have minimal unwanted side effects. Its intrinsicestrogenic activity should compensate for the loss of estrogen secretionby the ovaries after menopause, an effect not achievable by DHEA. Theinvention is useful for many diseases wherein activation of the estrogenreceptor will have beneficial effects, especially osteoporosis andmenopausal symptoms, including vaginal atrophy, insomnia, irritability,cardiovascular disease, urinary incontinence, and loss of libido. Inaddition, the invention is useful for treating and preventing diseaseswhich are responsible to the activation of the androgen receptor, e.g.bone loss, obesity, breast cancer, endometrial cancer, ovarian cancer,urinary incontinence, hypogonadism, loss of libido, loss or muscle mass,loss of energy, insulin resistance and other aging processes. Further,5-DIOL can be used to treat or prevent any condition which respondsfavorably to an improvement in the overall balance of circulating sexsteroids, namely estrogens and androgens.

Conditions expected to respond to the treatments herein may be diagnosedin conventional ways. For example, the appearance of breast cancer isusually detected by self breast examination, clinical breast examinationby the physician and/or mammography. Endometrial cancer, on the otherhand, is usually diagnosed by endometrial biopsy. Both cancers can bediagnosed and evaluated by standard physical methods well known to thoseskilled in the art, e.g. bone scan, chest X-Ray, skeletal survey,ultrasonography of the liver and liver scan (if needed), CAT scan, MRIand physical examination.

The onset of menopause is generally first recognized by the occurrenceof hot flashes. Further characterization of the menopause can bedetermined in accordance with known techniques. See for Example, TheMenopause (Herbert J. Buchsbaurm, ed), Springer Verlag, New York (1983),pp. 222. Vaginal atrophy is often indicated by dyspareunia and vaginalinfections. Vaginal atrophy, hypogonadism, diminished libido, insomnia,irritability, depression, and urinary incontinence are all characterizedin well-known ways. For the above-indicated disease, see, for example,Korenman, Stanley G. “Sexual Dysfunctions” in Williams Textbook ofEndocrinology, Jean D. Wilson and Daniel W. Foster, eds), WB SaundersCo., Philadelphia, pp. 1033-1048, 1992.

Bone density, on the other hand, can be measured by standard methodswell known to those skilled in the art, e.g. QDR (Quantitative DigitalRadiography), dual photon absorptiometry and computerized tomography.Plasma and urinary calcium and phosphate levels, plasma alkalinephosphatase, osteocalcin, calcitonin and parathormone concentrations, aswell as urinary hydroxyproline, deoxypyrrolidine, and calcium/creatininratios are useful parameters of bone formation and resorption.

Loss of collagen or connective tissues in the skin often accompaniesaging, especially in persons over 50 years of age. It may be evidencedby wrinkling of the skin and/or low elasticity. Skin status can beassessed by visual inspection, palpation and, with more precision, bypunch biopsy and standard histological examination.

The normal range of body weight is well known to those skilled in theart, while cholesterol and lipoproteins are routinely measured bystandard techniques (Nestler et al. J. Clin. Endocrinol. Metab. 66:57-61, 1988 for references).

In addition, 5-DIOL is useful as a female contraceptive. In the priorart, female contraception usually involves administering an estrogen,which at increased circulating levels, reduces LHRH secretion from thehypothalamus which, in turn, decreases LH secretion from the pituitary.The resultant reduction in LH secretion decreased ovarian function, andin particular ovulation. Addition of a progestin controlled the growthof the endometrium and transformed the vaginal and cervical secretionsinto an unfavorable environment or sperm capacitation and fertility.

In the present invention, 5-DIOL provides estrogen for contraceptionwhile simultaneously and desirably providing minimally increased levelsof androgens which will contribute to contraception since androgens alsoinhibit LHRH and LH secretion. These androgens can, especially in womenat perimenopause (as well as in postmenopausal women when contraceptionis no longer required), provide much needed stimulation of boneformation and resistance to bone loss. In addition to being a weakestrogen by itself, the estrogens produced from the administered 5-DIOLalso contribute to reducing bone loss. As with other uses discussedherein, use of 5-DIOL instead of a sex steroid (here estrogen) avoidsexternally administering relatively high doses of estrogens and thisavoids giving such estrogens extensive access to all tissues, many ofwhich do not require estrogens. By substituting 5-DIOL, estrogens and/orandrogens are instead produced by natural processes in the same tissueswhere estrogens and/or androgens are needed and that normally convert5-DIOL to estrogens and/or androgens. The relative proportions ofestrogen and androgen also remain substantially at natural levels ineach specific tissue.

Because ovarian function is diminished by the contraception techniquedescribed herein, the ovarian production of estrogen and progesteronewill be decreased. Thus, a progestin (e.g. medroxyprogesterone acetate,megestrol acetate, norethynodrel, L-norgestrel) may be administered aspart of the contraceptive method to prevent endometrial hypertrophy whenhigh doses of 5-DIOL are needed. The progestin may be administered in apharmaceutical composition tat includes the 5-DIOL or separately. Incertain embodiments, the progestin may be administered intermittentlyevery month for 12-14 days, or 12-14 days every few months (e.g. every2-5 months) or continuously, depending upon the dose of 5-DIOL usedwhich may well have no stimulatory effect on the endometrium atphysiological dose. Progestin dosage may be in the range utilized in theprior art but is preferably lower for reasons explained below.

Since 5-DIOL is converted to estrogen in many tissues, it is unlikelythat estrogen will need to be added to the contraceptive therapy tocompensate for the decreased estrogen production in the ovaries.However, minimum doses can be given, if necessary. Preferred dosage ofadded estrogen, when used in the contraceptive method is an amounteffective to achieve between 50 and 300 nanograms estradiol per liter orequivalent. Preferably the ratio of added estradiol to 5-DIOL (w/w) willrange from 100:1 to 10,000:1, preferably, 200:1 to 5,000:1 andespecially 300:1 to 3000:1. As with added progestin, added estrogen maybe administered as part of a pharmaceutical composition that includesthe 5-DIOL (or, where used, a prodrug of 5-DIOL) or separately.

In some embodiments, 5-DIOL, progestin and estrogen are alladministered, together or separately, as part of a combination therapy.A combination therapy results whenever a regimen of treatment elevatesblood levels of each active agent simultaneously. This simply requiresthat the active agents be administered sufficiently close in time thatelevated blood levels of these agents are concurrent.

The use of combination contraceptives containing estrogens andprogestins has not been shown to reduce the risk of breast cancer(Romiev et al., 1990, Cancer 66: 2253-2263). These data are consistentwith a known mitogenic effect of both estrogen and progesterone onbreast cell epithelial proliferation, thus explaining a peak of cellproliferation at mid-luteal phase (Masters et al., J. Natl. Cancer Inst.1977, 58: 1263-1265; Anderson et al., 1982, Brit. J. Cancer 46 376-382).In fact, total breast cell proliferation rate in premenopausal womenusing contraceptives is not different from that of untreated cyclingwomen (Potter et al., 1988; Brit. J. Cancer 58: 163-170; Going et al.,1988; Am. J. Pathol. 130: 193-204). The androgenic component of 5-DIOLshould reduce this potential harmful effect of estrogens and progestins.

Osteoblasts (bone forming cells) contain the enzymes which convert5-DIOL to estrogens and androgens. Therefore, 5-DIOL can be used insteadof (or in addition to) androgen, estrogen, or DHEA in the treatment orprevention of osteoporosis. Sufficient quantities of androgens areproduced in the bone (by conversion of the administered 5-DIOL) tostimulate bone formation and reduce bone loss. Furthermore, the lowestrogenic activity of 5-DIOL and the estrogens produced from theadministered 5-DIOL contribute to reducing bone loss.

Since bone mass density has been shown to be stimulated at particularlylow doses of androgens, 5-DIOL, which has a lower ratio ofandrogenic/estrogenic activity than DHEA should be able to have maximalbeneficial effects on the bone at a dosage which produces minimal risksof hyperandrogenism.

In addition, since 5-DIOL (or prodrugs thereof, if desired) istransformed to androgens and estrogens only by natural mechanismsexclusively in tissues that normally perform such transformationaccording to there local needs, side effects are greatly reduced oreliminated relative to externally administered active sex steroids ofthe prior art which have access to many tissues that neither produce norrequire a given androgen or estrogen. The physiological balance of sexsteroids n those tissues are thus not disturbed in accordance with thepresent invention, contrary to all hormone replacement therapies ofprior art. The relative ratio of androgens and estrogens produced fromthe 5-DIOL is also a substantially normal ratio instead of being anabnormally elevated ratio of one type of sex steroid as occurs when thatactive sex steroid is directly administered exogenously, thus causingexposure of all tissues, including those having no need for suchtherapy.

In one preferred treatment for menopausal symptoms, the invention seeksto simultaneously maintain blood levels of 5-DIOL, androgens, andestrogens within normal premenopausal parameters. Without intending tobe bound by theory, it is believed that maintenance of appropriateprecursor levels will better enable natural enzymes, such as17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid hydrogenase,aromatase and 5-reductase to regulate production of androgens andestrogens and to maintain them in a manner more closely resembling theirabsolute and relative levels prevailing prior to menopause. Hence, theinvention contemplates that not only estrogens but also androgens willbe kept in better balance. In fact, the target tissues possess theenzymatic machinery necessary to synthesize and inactivate androgensand/or estrogens according to local needs (Labrie, Mol. Cell.Endocrinol. 78, C113-C118, 1991).

As discussed above, 5-DIOL can be administered with estrogens. However,since compared to DHEA, a relatively higher proportion of estrogens thanandrogens are produced and 5-DIOL is itself a weak estrogen, it shouldbe possible to attain he desired level of estrogens without the additionof estrogens and without producing unwanted androgenic side effectsaccompanying high levels of DHEA, a more androgenic compound. Similarly,since 5-DIOL is a weak estrogen, progestin therapy may not be required.

However, if it is determined that additional estrogens are needed, theestrogen and 5-DIOL may be administered simultaneously or separately. Inaddition, it is necessary only that both the 5-DIOL and estrogen beadministered in a manner and at a dosage sufficient to allow blood serumconcentration of each to obtain desired levels. in accordance with thecombination therapy of the invention, concentration of the 5-DIOL ismaintained within desired parameters at the same time that estrogenconcentration is maintained within desired parameters. Where estradiolis used, serum estradiol concentration should typically be maintainedbetween 50 and 200 nanograms per liter, preferably between 100 and 175nanograms per liter and most preferably between 125 and 175 nanogramsper liter. Where another estrogen is used, serum concentration may bevaried in a known manner to account for the known difference inestrogenic activity relative to estradiol and in order to achieve normalpremenopausal estrogen levels. A lesser concentration is needed, forexample, if Mestranol is used. Adequate serum estrogen levels can alsobe assessed by disappearance of the symptoms of menopause. Serumconcentration of the 5-DIOL is typically maintained between 4 and 10 nMor women and between 10 and 20 nM for men or in some embodiments between4.0 and 7.0 nM for women or between 7.0 and 15 nM for men.

If estrogen is combined with 5-DIOL, it is preferably estradiol, but maybe estrone sulfate or another compound which acts as an estrogenreceptor agonist directly or following proper conversion. Whenadministered separately, commercially available estrogen supplements maybe used, e.g., PREMARIN, available from Ayerst (St. Laurent, Québec,Canada). For typical patients, the appropriate dosage of estrogen toachieve desired serum concentrations is between 0.3 and 2.5 milligramsof PREMARIN per day per 50 kg of body weight when administered orally.In certain embodiments of the invention, the estrogen may be17β-estradiol administered percutaneously in a patch which is availablefrom CIBA under the name ESTRADERM wherein the daily doses is between0.05 and 0.2 milligrams per day per 50 kg of body weight. For typicalpatients, the appropriate dosage of the sex steroid precursor 5-DIOL toachieve desired serum concentration of the precursor is between 0.10 and2.5 grams per day per 50 kg of body weight when administered orally.Other prodrugs will be administered at a dosage that depends on their invivo conversion rate to 5-DIOL. 5-DIOL may also be administeredtransdermally or transmucosally, as described in detail above, in asufficient amount to achieve target serum concentration.

In another embodiment, menopause is treated with 5-DIOL as set forthabove, in combination with periodic administration of a progestin suchas medroxyprogesterone acetate (e.g., Provera) which is preferablyadministered intermittently, e.g. at a dosage of 2-10 mg per day for 12consecutive days, said 12-day periods being spaced 20 days to 5 monthsapart. A combination therapy using 5-DIOL, an estrogen and a progestinmay also be used, preferably at the dosages discussed herein for eachcomponent.

The same doses of 5-DIOL will be used for all indications, exceptcontraception and prevention of ovarian and endometrial cancer whereserum levels of about to 15 nM 5-DIOL will be preferred.

For all other indications when androgens and/or estrogens are needed,the usual dosage mentioned above will be used. Similarly, when used incombination with an antiestrogen for the treatment or prevention ofbreast cancer, endometriosis, as other estrogen-sensitive disease, thesame dose of 5-DIOL will be used. In some cases, where an aromataseinhibitor, an inhibitor of 17β-hydroxysteroid dehydrogenase, anandrogen, a progestin, an inhibitor of gonadal steroid formation, anLHRH agonist or antagonist is used, the same dose of 5-DIOL isrecommended.

The following examples demonstrate the androgenic and estrogenic effectsof 5-DIOL and provide a comparison of the relative activities of 5-DIOLand DHEA. As discussed above, the preferential estrogenic activityrelative to androgenic activity of 5-DIOL compared to DHEA is shown.

Materials and Methods

Animals

Male and female Sprague-Dawley rats [Crl:CD(SD)Br] weighing 225-250 gand 175-200 g, respectively, were obtained from Charles River CanadaInc. (St-Constant, Québec) and housed 2 per cage under a regimen of 14 hof light/day (lights on at 07:15 h). Animals received Purina rat chowand water ad libitum. The animal studies were conducted to the“Guideline for Care and Use of Experimental Animals”.

Treatment

The animals were randomly divided into the indicated groups (8 and 10rats per group for subcutaneous and topical administration,respectively). The animals of the appropriate groups were bilaterallyovariectomized (OVX) or orchiectomized (ORCH) under ether anesthesiawhile other rats were used as intact controls. DHEA and 5-DIOL obtainedfrom Steraloids (Wilton, N.H., USA) were dissolved in 50% ethanol—50%propylene glycol and applied twice daily (0.5 ml) on the dorsal skinarea (2 cm×2 cm) for 7 days starting on the day of OVX or ORCH. Forsubcutaneous administration, DHEA and 5-DIOL were dissolved in 0.5 ml10% ethanol—1% gelatin—0.9% NaCl and injected twice daily in the dorsalarea for 7 days starting also on the day of OVX or ORCH.

Seven days after starting treatment or approximately 12 h after lastadministration of the steroid, the animals were killed by decapitation.Blood samples were collected individually and serum was frozen at −20°C. until assayed. Uteri, ovaries, ventral prostates, dorsal prostatesand seminal vesicles were immediately removed, freed from connective andadipose tissue, weighed, frozen in liquid nitrogen, and stored at −80°C. until assayed. Three rats from the indicated treatment groups wereperfused with paraformaldehyde for in situ hybridization.

Steroid Analysis

Steroid extraction. Ethanol (5 ml) was added to 1 ml serum andcentrifugation at 2000×g was performed for 15 min. The resulting pelletwas further extracted with 2 ml ethanol and, after a secondcentrifugation at 2000×g for 15 min, the two supernatants were combined.The suspension was recentrifuged as described above and the supernatantwas decanted and combined with the previously obtained ethanol extracts.The organic solvent was then evaporated under nitrogen and the residuewas dissolved in 1 ml water:methanol (95:5, v/v). The C-18 columns(Bound-Elut, Amersham, Bucks, U.K.) were conditioned by passingconsecutively 10 ml methanol, 10 ml water and 10 ml methanol/water(5:95, v/v). The extracts solubilized in water:methanol (95:5, v/v) werethen deposited on the C-18 columns. After washing the columns with 2 mlwater:methanol (95:5, v/v) and 5 ml methanol:water (50:50, v/v), 5 mlmethanol:water (85:15, v/v) were added to equate the non-conjugatedsteroids.

Chromatography on LH-20 Columns and Radioimmunoassay

Chromatography on Sephadex LH-20 columns (Pharmacia, Uppsala, Sweden)was performed as previously described (Bélanger et al., 1988). In brief,steroids were solubilized in 1 ml isooctane/toluene/methanol (90:5:5,v:v:v) and deposited on the LH-20 columns. Fractions were collected and,after evaporation of the organic solvent, the concentration of thevarious steroids was determined by radioimmunoassay as previouslydescribed (Bélanger et al., 1980; Bélanger et al., 1988; Bélanger etal., 1990).

Preparation of the cDNA Probes

The plasmid containing the DNA fragment complementary to the mRNAencoding PBP-C1 was kindly provided by Dr. Malcolm G. Parker (ImperialCancer Research Fund, London, United Kingdom). The 434-basepair Pst-Irestriction fragment of the PBP-C1 cDNA was purified by electroelution(Bio-Rad electro-eluter, model 422, Bio-Rad, Richmond, Calif.) afterelectrophoresis on a 5% (wt/vol) polyacrylamide gel. The purifiedfragment was radiolabeled with [a-³⁵S]dCTPaS (Amersham, Oakville,Ontario, Canada) to high specific activity (10⁹ dpm/μg) by the randomprimer method (Feinberg and Vogelstein, 1983).

Measurement of PBP-C1 mRNA Levels by in situ Hybridization

In situ hybridization of prostatic sections with the PBP-C1 probe wasperformed as described previously (Pelletier et al., 1988). In brief,rats were perfused with fixation buffer consisting of 4%paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The ventralprostates freed from fat and connective tissue, were postfixed infixation buffer for 2 h at 4 C. and subsequently soaked in 0.05 M PBScontaining 15% (wt/vol) sucrose. Thereafter, the ventral prostates wererapidly frozen in isopentane chilled in liquid nitrogen. Multiple (sixto eight) 10 μM tissue sections from each ventral prostate were mountedon gelatin-coated glass slides. Prehybridization buffer contained 50%formamide, 5×SSPE (1×SSPE=0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA, pH7.4), 0.1% sodium dodecyl sulfate, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.2 mg/ml yeast tRNA, 0.2 mg/ml denatured salmon testisDNA, and 2 μg/ml poly(A). The slides were hybridized in prehybridizationbuffer containing, in addition, 4% dextran sulfate and saturatingconcentrations (1.0-1.5×10⁷ cpm/ml) of the PBP-C1 cDNA probe for 48 h at37 C. The sections were then washed twice for 30 min in 2×SSC(1×SSC=0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), dehydrated, andexposed for autoradiography. To determine the amount of nonspecificbackground hybridization, prostatic sections for each treatment groupwere treated with pancreatin ribonuclease-A (20 μg/ml) for 1 h at roomtemperature before prehybridizing sections from rat brain, pituitary,kidney, and liver with the PBP-C1 probe. No specific hybridizazion couldbe observed (data not shown)

LH Radioimmunoassay

Serum LH was measured by double-antibody radioimmunoassay using rathormones (LH-I-6 for iodination; LH-RP-2 as standard), and the rabbitantisera anti-r-LH-S-8, generously supplied by the National PituitaryProgram, Baltimore, USA.

Statistical Analyses

Radioimmunoassay data were analyzed using a program based on model II ofRodbard and Lewald (Rodbard, 1974). Plasma steroid levels are shown asthe means±SEM of duplicate determinations of individual samples. In situhybridization data were obtained as follows: for each prostatic tissuesection, 20 randomly selected areas measuring 0.25 mm² (excluding acinarlumen) were analyzed using an Image Research Analysis System (Amersham,Arlington Heights, Ill.), and the mean optical density value for eachsection was calculated. Data are shown as the means±SEM of 20 readingsfrom 6-8 prostatic sections originating from ventral 3 prostates.Statistical significance was measured according to the multiple rangetest of Duncan-Kramer (Kramer, 1956).

As shown in FIG. 1, DHEA and 5-DIOL produced similar stimulatory effectson uterine weight, an indicator of estrogenic activity. In particular,the administration of the 10 mg and 30 mg doses of DHEA and 5-DIOLproduced a comparable stimulation of uterine weight in theovariectomized rat, while 1 and 3 mg doses had no significant effect.However, at 100 mg, the highest dose used, 5-DIOL produced a greaterstimulatory effect with a maximal 52% reversal of the inhibitory effectof the ovariectomy as compared to the maximal 42% reversal produced byDHEA.

The effects of 5-DIOL and DHEA on serum LH are shown in FIG. 2. Serum LHis known to be a sensitive indicator of both androgenic and estrogenicactivity based on the findings that serum LH increases rapidly upon theremoval of the predominant inhibitory feedback action of sex steroidsafter gonadectomy in male as well as female animals (Ferland et al., In:Labrie, F., Meites, J., and Pelletier, G. (eds), Hypothalamus andEndocrine Functions, pp. 191-209. New York: Plenum Press, 1976). At the10 mg dose, 5-DIOL produced greater effects and completely reversed thepotent stimulatory effect of ovariectomy to 0.86±0.17 ng/ml while thecorresponding dose of DHEA only caused a 61% (p<0.01) inhibitory effect.However, at higher doses, DHEA and 5-DIOL were found to have similareffects, with the 30 mg and 100 mg doses, respectively, inhibiting serumLH levels by approximately 35% and 70% below the value found in intactcontrol animals.

The effects on the circulating levels of the primary steroids andprecursors produced by the administration of 5-DIOL and DHEA are shownin FIGS. 3 through 7. In particular, as shown in FIG. 3, treatment withDHEA caused serum DHEA to increase from undetectable levels in controlovarectomized animals to 1.74±0.30 nM (p<0.01), 3.67±0.59 nM (p<0.01),12.9±3.69 nM (p<0.01) and 39.2±6.5 nM (p<0.01) after administration ofthe 3, 10, 30 and 100 mg dosages, respectively. On the other hand, arelatively constant but low level of serum DHEA was produced with thesame dosages of 5-DIOL.

FIG. 4 shows the serum levels of 5-DIOL which are produced afteradministration with 5-DIOL and DHEA. While at the lower doses, 1 and 3mg, 5-DIOL produced higher levels of 5-DIOL, at the higher doses, therewas no significant difference in the levels of 5-DIOL produced by 5-DIOLand DHEA.

The serum concentrations of 4-dione, testosterone anddihydrotestosterone (DHT) produced after administration of DHEA and5-DIOL are shown in FIGS. 5 through 7. The levels of serum 4-dione, anandrogenic indicator, produced by DHEA, ranged, depending on the dosage,from 30% to 125% higher than produced by 5-DIOL.

Consistent results were obtained with serum DHT and testosterone, bothindicators of androgenic activity. Specifically, DHEA produced 30% to125% higher serum DHT levels than 5-DIOL and at the highest dose, DHEAproduced a 53% greater stimulatory effect on testosterone than the samedose of 5-DIOL. Furthermore, the levels of serum DHT produced by 5-DIOLremained substantially constant and relatively low across the dosagerange.

FIGS. 8 through 11 are directed to the effects of DHEA and 5-DIOL on avariety of well-recognized androgen-sensitive Parameters in theorchiectomized rat. FIG. 3 shows the effects on ventral prostate weight,wherein at the 10 mg dose, DHEA was able to reverse by about 75% theinhibitory effect of orchiectomy while a 150% higher dose (15 mg) of5-DIOL was only able to produce a 50% reversal of the effect ofcastration. In addition, DHEA was 1-fold more potent in increasingseminal vesicle weight, as shown in FIG. 9.

Furthermore, as shown in FIGS. 10 and 11, DHEA produced two to fivetimes the effect produced by 5-DIOL on the concentration of the mRNAsencoding the C1 and C3 components of prostate binding protein (PBP)which, as discussed above, are precise indicators of androgenicactivity. Moreover, the maximal levels of C1 and C3 PBP mRNAs achievedwith the highest doses of 5-DIOL were only 17% to 37% of the levelsobtained by DHEA.

FIGS. 12 through 16 show the effects of subcutaneous administration ofDHEA and 5-DIOL on selected parameters described above. Since the twosteroids were injected subcutaneously, these results provide a moredirect measure of the relative estrogenic and androgenic activities ofDHEA and 5-DIOL under conditions of optimal bioavailability. Inparticular, as shown in FIG. 12, 5-DIOL, at the lowest dose used, namely0.3 mg, reversed by 90% the effect of 1-week ovariectomy on uterineweight (an estrogen-sensitive parameter) while DHEA, at the same dose,had no significant effect. However, at the higher dosages, the maximalstimulatory effect achieved by the two steroids was similar with a 2.8to 2.9-fold stimulation. Furthermore, as calculated from the dosesgiving half-maximal stimulation of uterine weight (˜2.5 mg for DHEA and0.5 mg for 5-DIOL), 5-DIOL is approximately 5.0 times more uterotrophicthan DHEA following subcutaneous administration. In addition, as shownin FIG. 13, 5-DIOL was found to be 5- to 7-fold more potent than DHEA ininhibiting the elevated serum LH levels indicated by castration.

As shown in FIG. 14, the maximal stimulatory effects on prostate andseminal vesicle weights (androgen-sensitive parameters) obtained with5-DIOL were about 70% of the values achieved with DHEA. However, ascalculated from the doses giving half-maximal reversal of the effect oforchiectomy (ED₅₀), DHEA and 5-DIOL had approximately an equal potency(i.e. an ED₅₀ value of 1 mg). On the other hand, the ED₅₀ values ofmaximal DHEA and 5-DIOL effects were calculated at 2.5 mg and 1.2 mg,respectively, for an estimated 2-fold higher potency of 5-DIOL comparedto DHEA. Similarly, as shown in FIG. 15, for seminal vesicle weight, themaximal stimulatory effect of 5-DIOL was approximately 70% that of DHEAwith half-maximal reversals of the effect of orchiectomy estimated at2.5 and 1.2 mg, respectively, for DHEA and 5-DIOL for an estimated2-fold high potency of 5-DIOL. On the prostatic concentrations of C1 andC3 PBP mRNAs, 5-DIOL was about twice as potent as DHEA. FIG. 16 showsthe effects on serum LH concentration, with 5-DIOL being approximately10 times more potent than DHEA.

From the foregoing, it may be seen that, at every concentration, theratio of estrogenic to androgenic effects provided by DHEA is moreheavily weighted toward androgenic effects than is that ratio for5-DIOL.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will be apparent to those skilled in the art.

What is claimed is:
 1. A method for treating or reducing the likelihoodof acquiring skin atrophy, comprising the step of administering atherapeutically effective amount of androst-5-ene-3β,17β-diol or prodrugto a patient in need thereof.
 2. A method for treating or reducing thelikelihood of acquiring skin dryness, comprising the step ofadministering a therapeutically effective amount ofandrost-5-ene-3β,17β-diol or prodrug to a patient in need thereof.